DNA实验技术

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

Mutagenesis by PCR(adapted from Ito et al Gene 102 67-70)This method needs only a single mutagenic primer (+ 3 other primers which are the same for all constructs if used in the same vector) and woul ...

QuikChange (Stratagene)This is a quick and reliable (and dead easy too!) method for PCR but costs more. It is however highly recommended if you need to make only a small number of mutants or if you ca ...

Preparation of Nested DeletionsProcedure:1) Need to cut 10 µg of plasmid in two spots ('A' and 'B') in polylinker. 'A'cut is 'ith a restriction enzyme which gives a 3' recessed end (exonuclease sensit ...

Generation of uni-directional nested deletions in plasmid DNA1) Digest DNA with two restriction enzymes such that one end is ExoIII susceptible (blunt or 5' overhang) and the other is ExoIII resistant ...

Site Directed Mutagenesis- (Kunkel Method) 1. Phosphorylate mutagenic oligonucleotide. 6 uL Primer 1 uL 20 mM ATP 1 uL 10X T4 Kinase buffer 2 uL T4 Kinase (20 units)Incubate at 37oC for 45 min.2. Sec ...

In vitro Mutagenesis with dut ung single stranded DNASteve Hahnlast modified 3/3/00Kinase Oligonucleotide:(gel purified oligonucleotides give highest mutation frequency)0.5 microgram oligonucleotide2 ...

OverviewTargeted Amplification of Mutant Strand (TAMS) technology allows multiple-site mutagenesis in a simple half-day protocol. It’s the only method that canmutate first strand cDNA directly and pe ...

Question ...

Desalting Oligonucleotides by Gel FiltrationGeneralDesalting or buffer exchange on a gel filtration column is dependent on volume not quantity of DNA in solution. Many sizes of gel filtration columns ...

Recommended Storage Conditions Probes should be stored frozen both when lyopholized or in solution. Aliquoting your probe into additional vials after resuspension can help to minimize potential breakd ...

General Design GuidelinesThe following guidelines should be taken into account when designing modified oligonucleotides. Sequence Length - SYNTHEGEN can synthesize oligonucleotides from 5 to 110 bases ...

ROX StandardThe final working concentration of ROX Standard in the PCR buffer is 0.5 µMolar (1X). SYNTHEGEN's ROX Standards are shipped in liquid form at a concentration of 100 µMolar (200X). ...

1. Prior to precipitation dilute DNA solution may be concentrated to allow precipitation in smaller volumes.2. Add an equal volume of 2-Butanol to DNA solution and mix by vortexing. 3. Centrifuge samp ...

1. If the trityl group is not removed from a newly-synthesized oligonucleotide it may interfere with polymerization reactions.2. Evaporate the oligonucleotide to dryness.3. Add 300 ml 80% Acetic acid. ...

1. Pour and polymerize a 20% polyacrylamide gel no Urea.2. Remove clamps. Rinse with water. Remove comb. Rinse top of gel well.3. Insert comb teeth down until teeth just touch the gel. Mount the plate ...

How to Calculate the Melting Temperature of Oligonucleotides The melting temperature Tm of an oligonucleotide is its most critically important value. The most reliable and accurate determination of me ...

UV Quantitation of DNA DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily quantitated in a UV spectrometer. Typically 1 OD260 (i.e. a solution having an absorban ...